EP2813223B1 - Agents d'apport métallique et leur utilisation thérapeutique - Google Patents

Agents d'apport métallique et leur utilisation thérapeutique Download PDF

Info

Publication number
EP2813223B1
EP2813223B1 EP14166833.5A EP14166833A EP2813223B1 EP 2813223 B1 EP2813223 B1 EP 2813223B1 EP 14166833 A EP14166833 A EP 14166833A EP 2813223 B1 EP2813223 B1 EP 2813223B1
Authority
EP
European Patent Office
Prior art keywords
metal
complexes
cells
disease
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP14166833.5A
Other languages
German (de)
English (en)
Other versions
EP2813223B8 (fr
EP2813223A1 (fr
Inventor
Kevin Jeffrey Barnham
Paul Stephen Donnelly
Anthony Robert White
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Melbourne
Original Assignee
University of Melbourne
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Melbourne filed Critical University of Melbourne
Priority to EP17170883.7A priority Critical patent/EP3238719A1/fr
Publication of EP2813223A1 publication Critical patent/EP2813223A1/fr
Publication of EP2813223B1 publication Critical patent/EP2813223B1/fr
Application granted granted Critical
Publication of EP2813223B8 publication Critical patent/EP2813223B8/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/30—Copper compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic Table
    • C07F1/08—Copper compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F3/00—Compounds containing elements of Groups 2 or 12 of the Periodic Table
    • C07F3/06—Zinc compounds

Definitions

  • the present invention relates to the use of metal complexes as pharmaceutical or veterinary agents, in particular for the treatment of conditions in which metal delivery can prevent, alleviate or ameliorate the condition.
  • metal complexes as pharmaceutical or veterinary agents, in particular for the treatment of conditions in which metal delivery can prevent, alleviate or ameliorate the condition.
  • metal delivery can prevent, alleviate or ameliorate the condition.
  • the life span is thought to be biologically fixed for each species, and the length of the human life span is uncertain, but may be up to 120 years. Since life expectancy has risen significantly in this century, the elderly are an increasing segment of our population, and their health care needs will continue to grow for decades.
  • Metalloenzymes of this type are involved in a number of important bio catalytic processes including reduction of excess oxygen species. Accordingly whenever there is either too high or too low a level of metals present in a biological system either too high a level or too low a level the normal biological processes are interrupted, typically leading to undesirable consequences. This typically occurs as many of the crucial enzymatic processes that provide protection in the biological system are suppressed or inactivated leading to undesirable consequences.
  • oxidative stress which is related to abnormal metal levels as many of the protective enzymes responsible for alleviating oxidative stress are deactivated if biological metal levels are too low.
  • OS oxidative stress
  • Other conditions associated with OS include cancer, cataracts, neurodegenerative disorders such as Alzheimer's disease and heart diseases.
  • OS plays a prominent role in three types of neuromuscular disorders: amyotrophic lateral sclerosis (ALS), mitochondrial/metabolic disease and Friedreich's ataxia.
  • ALS amyotrophic lateral sclerosis
  • mitochondrial/metabolic disease mitochondrial/metabolic disease
  • the effect of OS is not limited to any one part of the human body, with examples of the negative effects of OS being observed for almost all organs.
  • the human brain is an organ that concentrates metal ions and recent evidence suggests that a breakdown in metal homeostasis plays a critical role in a variety of age-related neurodegenerative diseases. Common features of these diseases include the deposition of misfolded protein (each disease can have its own specific amyloid protein) and substantial cellular damage as a result of OS.
  • OS is the primary cause of physical damage in a wide range of disease states, including amyloidogenic neurological disorders such as Alzheimer's disease (AD), amylotrophic lateral sclerosis (ALS), prion diseases - including Creutzfeldt-Jakob Disease (CJD), transmissible spongioform encephalopathies (TSE), cataracts, mitochondrial disorders, Menke's disease, Parkinson's disease (PD) and Huntington's disease (HD).
  • AD Alzheimer's disease
  • ALS amylotrophic lateral sclerosis
  • CJD Creutzfeldt-Jakob Disease
  • TSE transmissible spongioform encephalopathies
  • cataracts mitochondrial disorders
  • Menke's disease Menke's disease
  • Parkinson's disease Parkinson's disease
  • HD Huntington's disease
  • Copper metal ion deficiency has been reported as a condition associated with AD. Copper is an essential element that is required for many enzymes to function properly, particularly those enzymes that maintain a balance in antioxidant/pro-oxidant homeostasis such as superoxide dismutase and cytochrome C oxidase.
  • One consequence of copper deficiency is that the protective enzymes responsible for detoxifying reactive oxygen species (ROS) are inadequately loaded with copper and therefore do not effectively carry out normal enzyme function.
  • ROS reactive oxygen species
  • the inadequate loading of such protective enzymes leads to a general increase in OS (as is observed in AD) which will be reflected in increased protein oxidation, such as increased protein carbonyls.
  • vitamin E was found to be ineffective at decreasing the oxidative stress at the substantia nigra (The Parkinson Study Group, 1993, Offen et al., 1996) since this compound, although capable of crossing the blood brain barrier, is trapped in the cell membrane and therefore does not reach the cytoplasm where its antioxidant properties are needed. Vitamin C also does not cross the blood brain barrier and therefore, cannot be used effectively for neurodegenerative diseases of central origin.
  • the present invention is therefore based on the finding that certain metal complexes are effective in delivering bio-available metal and could thus be used in the treatment of conditions which can be prevented, treated or ameliorated by metal delivery.
  • the metal be released in the cell such that after metal delivery the metal is present in the form of the free cation and it is the free cation that leads to the observed biological activity.
  • the metal stay in the form of the bound complex even after metal delivery and with these conditions it is the bound form of the metal (the metal complex) that is biologically active in the cell.
  • Wada et al. Archives of Biochemistry and Biophysics, 310(1), 1-5 (1994 ), describes a stable superoxide dismutase-like copper complex with high membrane permeability.
  • the invention provides a metal complex of Formula (I) for use in the treatment of Parkinson's disease or amyotrophic lateral sclerosis: where:
  • R 2 and R 6 are each methyl.
  • R 2 and R 6 are each ethyl.
  • R 2 and R6 are each phenyl.
  • the disorder is Parkinson's disease. In a specific embodiment the disorder is Amyotrophic lateral sclerosis (ALS).
  • ALS Amyotrophic lateral sclerosis
  • a method of prophylaxis or treatment of oxidative stress including administering a therapeutically effective amount of a metal complex of formula (I) to the subject.
  • a metal complex of formula (I) in the preparation of a medicament for the treatment or prophylaxis of OS.
  • a method of protecting a cell from OS including exposing the cell to an effective amount of a metal complex of formula (I).
  • the cell is a cell in a subject and exposing the cell to the metal complex includes administering the metal complex to the subject.
  • a method of prophylaxis or treatment of a tau related disorder including administering a therapeutically effective amount of a metal complex of formula (I) to the subject.
  • the tau related disorder is a neurodegenerative disorder.
  • a metal complex of formula (I) in the preparation of a medicament for the treatment or prophylaxis of a tau related disorder.
  • a method of reducing or preventing the effects of Abeta on a cell including exposing the cell to an effective amount of a metal complex of the formula (I).
  • the cell is a cell in a subject and exposing the cell to the metal complex includes administering the metal complex to the subject.
  • a method of prophylaxis or treatment of an Abeta related disorder including administering a therapeutically effective amount of a metal complex of formula (I) to the subject.
  • kinase is a receptor tyrosine kinase.
  • the receptor tyrosine kinase is epidermal growth factor receptor (EGFR).
  • the kinase is selected from the group consisting of ERK, PI3K, Akt, GSK3 and JNK.
  • a common feature of the methods and uses as outlined above is the use of a metal complex of formula (I).
  • the metal complex is sufficiently stable that upon administration to the subject the metal is not released in the extracellular environment but rather is released in the cells of the subject. This is preferable as it ensures that the metal is delivered to the cells of the subject rather than being released prior to delivery to the cells.
  • the metal is released from the complex in the cell it is therefore present in the cell as the free cation and it is the free cation that is responsible for the biological activity in the subject.
  • the metal complex does not release the metal in the extracellular matrix nor does it release the metal in the cell rather it is the metal complex that leads to the observed biological activity. Modifications to the metal complex either through changes in the nature of the metal or changes in the nature of the ligand may be made to obtain the desired delivery of the metal to the cells of the subject.
  • the metal is Copper.
  • the complex is symmetrical.
  • R 1 is H.
  • R 2 is selected from the group consisting of methyl, ethyl, and phenyl.
  • R 3 is methyl
  • R 4 is methyl
  • R 5 is H.
  • R 6 is selected from the group consisting of methyl, ethyl, and phenyl.
  • the metal complexes described herein to increase phosphoinositol-3-kinase (PI3K) -Akt activity in the subject.
  • the metal complex decreases glycogen synthase kinase 3 (GSK3) activity in the subject.
  • the metal complex increases JNK activity in the subject.
  • the metal complex leads to activation of one or more anti-oxidant enzymes.
  • the anti-oxidant enzyme is superoxide dismutase (SOD).
  • Alkyl as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C 1 -C 14 alkyl, more preferably C 1 -C 1O alkyl, most preferably C 1 -C 6 unless otherwise noted.
  • suitable straight and branched C 1 -C 6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like.
  • alkyl When alkyl is used as a bridging group it is typically (but not exclusively) referred to as alkylene. A similar convention applies to other bridging groups.
  • acyl means an alkyl-CO- group in which the alkyl group is as described herein.
  • examples of acyl include acetyl and benzoyl.
  • the alkyl group is preferably a C 1 -C 6 alkyl group.
  • Alkenyl as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-14 carbon atoms, more preferably 2-12 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain.
  • the group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z.
  • Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.
  • Alkoxy refers to an -O-alkyl group in which alkyl is defined herein.
  • the alkoxy is a C 1 -C 6 alkoxy. Examples include, but are not limited to, methoxy and ethoxy.
  • Alkynyl as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-14 carbon atoms, more preferably 2-12 carbon atoms, more preferably 2-6 carbon atoms in the normal chain.
  • Exemplary structures include, but are not limited to, ethynyl and propynyl.
  • Cycloalkyl refers to a saturated or partially saturated, monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane.
  • Heterocycloalkyl refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered.
  • heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane.
  • Heteroalkyl refers to a straight- or branched-chain alkyl group preferably having from 2 to 14 carbons, more preferably 2 to 10 atoms in the chain, one or more of which is a heteroatom selected from S, O, and N.
  • exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl sulfides, and the like.
  • Aryl as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring.
  • aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C 5-7 cycloalkyl or C 5-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.
  • Heteroaryl either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur.
  • heteroaryl examples include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, 1H-indazo!e, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-
  • terapéuticaally effective amount or “effective amount” is an amount sufficient to effect beneficial or desired clinical results.
  • An effective amount can be administered in one or more administrations.
  • An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
  • treatment means affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect and include: (a) preventing the condition from occurring in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it; (b) inhibiting the condition, i.e., arresting its development; or (c) relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition.
  • subject refers to any animal having a disease or condition which requires treatment or prophylaxis with a biologically-active agent.
  • the subject may be a mammal, typically a human, or may be a non-human primate or non-primates such as used in animal model testing. While it is particularly contemplated that the compounds are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids and ungulates.
  • the present invention is based on the observation that metal plays an important role in a wide number of biological processes and adequate metal levels are especially important in the efficient functioning of a wide range of biologically important enzymes and cellular signaling processes.
  • Enzymes that involve metal activation include many of the enzymes related to oxidation at the cellular level. Accordingly it was felt that selective metal delivery to subjects with conditions relating to abnormal levels of metals could provide a useful therapeutic outcome for a number of biological applications. In particular it was felt that this could be useful in respect of conditions caused by or associated with oxidative stress as this is a condition where many of the protective mechanisms or enzymes that protect the body from oxidative stress involve metal catalysis and so the provision of bio-available metal may be a useful therapeutic in treating these conditions.
  • the cell permeable metal complex be sufficiently stable such that upon administration to a subject the metal is not released in the extra-cellular environment.
  • a further advantage of the use of metal complexes over the "naked" metal ion is that delivery of the metal can be targeted which reduces the chance that unwanted side effects will be observed (for example copper toxicity). A number of metal complexes meet these criteria.
  • metal complexes for use in the methods of the present invention are metal complexes of bis(thiosemicarbazone) (BTSC) ligands which have been investigated as metallodrugs and have proven to have a broad range of pharmacological activity.
  • BTSC bis(thiosemicarbazone)
  • BTSC ligands as vehicles for the selective delivery of radioactive copper isotopes to hypoxic tissue and leucocytes in the development of radiopharmaceuticals.
  • Cu(II) is reduced by intracellular reductants to Cu(I) which subsequently dissociates from the ligand.
  • Other Cu(II)-BTSC complexes are more resistant to reduction and disassociation, and are only trapped in hypoxic cells.
  • This selectivity is remarkably sensitive to the number of alkyl groups attached to the diimine backbone of the ligand.
  • copper(II)diacetylbis(N(4)-methylthiosemicarbazone) [Cu(ATSM)] with two methyl substituents on the backbone is selective for hypoxic cells whereas the copper of [Cu(GTSM)] is trapped in all cells.
  • hypoxic cell selectivity has been correlated with the Cu(II)/Cu(I) reduction potential, [Cu(ATSM)] is some 160 mV harder to reduce than [Cu(GTSM)], but differences in pKa and the stability of the reduced state may also be important.
  • [Zinc(BTSC)] complexes are also capable of transporting zinc into cells and a recent report used the intrinsic fluorescence of certain [Zn(BTSC)] complexes to probe the intracellular distribution of the complexes via fluorescence microscopy in several cancer-cell lines. Sub-cellular localization was a sensitive function of terminal nitrogen substituents on the complexes and cell type, varying from predominantly nucleolar to lysosomal. Zinc is central to a number of cell signal pathways including modulation of NMDA receptor activity expression of metallothienein and activation of mitogen activated protein kinase (MAPK)-mediated signal transduction pathways and therefore, Zn-BTSC uptake could have complex effects on downstream metal-mediated cell signalling.
  • MPK mitogen activated protein kinase
  • BTSC-metal complexes have several properties that make them worthy of investigation as potential therapeutic agents for the treatment of conditions related to oxidative stress including neurodegenerative disorders such as Alzheimer's disease. Organ and tissue distribution of these types of materials are well characterized, several BTSC complexes are known to be capable of crossing the blood brain barrier and there is no inherent class toxicity with these complexes. Importantly, the ligands can be readily modified by varying the nature and number of alkyl substituents on the ligand and these modifications can allow subtle control of subcellular targeting and metal release/retention properties.
  • the complexes are attractive as different metal complexes have different modes of metal release in the cell, potentially opening the way for the selective use of different metal complexes for different applications.
  • the zinc and copper complexes increase the bio-available metal via different mechanisms.
  • complexes of this type were attractive as not only do they have different mechanisms of metal release depending upon the metal ion chosen some of the complexes are such that they do not release the metal at all and so they may be used in circumstances where it is desirable not to deliver the metal in the form of a metal cation but rather it is desirable to deliver the metal in the form of a bound metal still in complex with the ligand.
  • the ligands and complexes selected for the intitial study were as follows: GTSM Cu(GTSM) Zn(GTSM) ATSMH 2 Cu(ATSM) Zn(ATSM) ATSEH 2 Cu(ATSE) Zn(ATSE) ATSPH 2 Cu(ATSP) Zn(ATSP) ChexTSEH 2 Cu(ChexTSE) Zn(ChexTSE)
  • Treatment of cells with [Zn(BTSC)] complexes resulted in significant increases in the intracellular Zinc levels as measured by ICP-MS ( Figure 2 .).
  • [Zn(ATSM)] and [Zn(ATSE)] induced 8.2 ⁇ 0.25 and 9.8 ⁇ 0.9 fold increases in cellular Zinc levels respectively ( Figure 2 ).
  • the data obtained suggested that the complexes were capable of delivering metals to cells and so attention was turned to probing a number of biological systems where it was envisaged that metal delivery could be useful
  • the lower stability of the Zn-BTSC complexes means they are more susceptible to intracellular transchelation than their Copper analogues and therefore, could elevate levels of bio-available Zinc within the cells.
  • the elevated Zinc levels in the cells treated with [Zn(BTSC)] complexes correlated with a reduction in the extracellular levels of A ⁇ 1-40.
  • the concentration of A ⁇ 1-40 in the medium of untreated cells was 0.6-0.8 ng mL -1 and was reduced to less than 0.2 ng mL -1 following treatment with 25 ⁇ M [Zn(BTSC)].
  • the different [Zn(BTSC)] complexes exhibited some detectable differences in the dose dependent reduction of A ⁇ 1-40.
  • the complexes of the invention have been shown to be effective as metal delivery agents, particularly agents for the delivery of metals to cells. According the complexes described herein may be used in the treatment or prophylaxis of a number of conditions in which metal delivery can prevent, alleviate or ameliorate the condition.
  • conditions of this type There are a number of conditions of this type.
  • An example of conditions of this type is conditions associated with or caused by oxidative stress. It is known that many of the protective biological anti-oxidant mechanisms involve metal catalysed enzymes and thus metal delivery can serve to stimulate or re-start the activity of the biological anti-oxidant mechanisms leading to an overall anti-oxidant effect being achieved.
  • the condition associated with or caused by oxidative stress is selected from the group consisting of cardiovascular conditions, cancers, cataracts, neurological disorders such as Alzheimer's disease, prion diseases - including Creutzfeldt-Jakob Disease (CJD), and heart diseases, amyloidogenic amylotrophic lateral sclerosis (ALS), prion transmissible spongioform encephalopathies (TSE), cataracts, mitochondrial disorders, Menke's disease, Parkinson's disease and Huntington's disease.
  • cardiovascular conditions such as Alzheimer's disease, prion diseases - including Creutzfeldt-Jakob Disease (CJD), and heart diseases, amyloidogenic amylotrophic lateral sclerosis (ALS), prion transmissible spongioform encephalopathies (TSE), cataracts, mitochondrial disorders, Menke's disease, Parkinson's disease and Huntington's disease.
  • CJD Creutzfeldt-Jakob Disease
  • TSE prion transmissible spongioform
  • the disorder is a neuromuscular disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS), mitochondrial/metabolic disease and Friedreich's ataxia.
  • ALS amyotrophic lateral sclerosis
  • mitochondrial/metabolic disease mitochondrial/metabolic disease
  • Friedreich's ataxia a neuromuscular disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS), mitochondrial/metabolic disease and Friedreich's ataxia.
  • condition is a neurological condition or a neurodegenerative disorder.
  • neurodegenerative condition is used herein in its broadest sense and refers to conditions in which various cell types of the nervous system are degenerated and/or have been damaged as a result of neurodegenerative disorders or injuries or exposures.
  • complexes of formula (I) can be used for the treatment of resulting conditions, in which damage to cells of the nervous system has occurred due to surgical interventions, infections, exposure to toxic agents, tumours, nutritional deficits or metabolic disorders.
  • the complex of formula (I) can be used for the treatment of the sequelae of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amylotrophic lateral sclerosis, epilepsy, drug abuse or drug addiction (alcohol, cocaine, heroin, amphetamine or the like), spinal cord disorders, dystrophy or degeneration of the neural retina (retinopathies) and peripheral neuropathies, such as diabetic neuropathy and/or the peripheral neuropathies induced by toxins.
  • neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amylotrophic lateral sclerosis, epilepsy, drug abuse or drug addiction (alcohol, cocaine, heroin, amphetamine or the like), spinal cord disorders, dystrophy or degeneration of the neural retina (retinopathies) and peripheral neuropathies, such as diabetic neuropathy and/or the peripheral neuropathies induced by toxins.
  • neuronal integrity can be threatened when neuronal cells display decreased survival or when the neurons can no longer propagate a signal.
  • Neurological conditions that can be treated with the complexes of the present invention include acute intermittent porphyria; adriamycin-induced cardiomyopathy; AIDS dementia and HIV-1 induced neurotoxicity; AD; ALS; atherosclerosis; cataract; cerebral ischaemia; cerebral palsy; cerebral tumour; chemotherapy-induced organ damage; cisplatin-induced nephrotoxicity; coronary artery bypass surgery; CJD and its new variant associated with "mad cow" disease; diabetic neuropathy; Down's syndrome; drowning; epilepsy and post-traumatic epilepsy; Friedrich's ataxia; frontotemporal dementia; glaucoma; glomerulopathy; haemochromatosis; haemodialysis; haemolysis; haemolytic uraemic syndrome (Weil's disease); Menke's disease; haemorrhagic stroke; Haller convinced-Spatz disease; heart attack and reperfusion injury; HD; Lewy body disease; intermittent claudication; ischaemic stroke;
  • complexes described herein may also be used to potentiate the effects of other treatments, for example to potentiate the neuroprotective effects of brain derived nerve growth factor.
  • the complexes described herein may also be used to treat Anemia, Neutropenia, Copper deficiency Myelopathy, Copper deficiency Syndrome and Hyperzincaemia.
  • treatments described herein are particularly directed to conditions which induce oxidative damage of the central nervous system, including acute and chronic neurological disorders such as, cerebral ischaemia, stroke (ischaemic and haemorragic), subharrachnoid haemorrage/cerebral vasospasm, cerebral tumour, AD, CJD and its new variant associated with "mad cow" disease, HD, PD, Friedrich's ataxia, cataract, dementia with Lewy body formation, multiple system atrophy, Haller Camill-Spatz disease, diffuse Lewy body disease, amylotrophic lateral sclerosis, motor neuron disease, multiple sclerosis, fatal familial insomnia, Gertsmann Straussler Sheinker disease and hereditary cerebral haemorrhage with amyoidoisis-Dutch type.
  • acute and chronic neurological disorders such as, cerebral ischaemia, stroke (ischaemic and haemorragic), subharrachnoid haemorrage/cerebral vasospasm, cerebral tumour, AD, CJD and
  • the neurodegenerative amyloidosis may be any condition in which neurological damage results from the deposition of amyloid.
  • the amyloid may be formed from a variety of protein or polypeptide precursors, including but not limited to A ⁇ , synuclein, huntingtin, or prion protein.
  • condition in one embodiment is selected from the group consisting of sporadic or familial AD, ALS, motor neuron disease, cataract, PD, Creutzfeldt-Jacob disease and its new variant associated with "mad cow” disease, HD, dementia with Lewy body formation, multiple system atrophy, Hallerêt-Spatz disease, and diffuse Lewy body disease.
  • the neurodegenerative amyloidosis is an A ⁇ -related condition, such as AD or dementia associated with Down syndrome or one of several forms of autosomal dominant forms of familial AD (reviewed in St George-Hyslop, 2000). Most preferably the A ⁇ -related condition is AD.
  • ADAS AD Assessment Scale
  • the complex and methods described herein may also be suitable for use in the treatment or prevention of neurodegenerative conditions, or may be suitable for use in alleviating the symptoms of neurodegenerative conditions. If administered to a subject who has been identified as having an increased risk of a predisposition to neurodegenerative conditions, or to a subject exhibiting pre-clinical manifestations of cognitive decline, such as Mild Cognitive Impairment or minimal progressive cognitive impairment, these methods and compounds may be able to prevent or delay the onset of clinical symptoms, in addition to the effect of slowing or reducing the rate of cognitive decline.
  • MCI Mild Cognitive Impairment
  • MCI can be detected using conventional cognitive screening tests, such as the Mini Mental Status Exam, and the Memory Impairment Screen, and neuropsychological screening batteries.
  • cancer Another condition that may be able to be treated by metal delivery is cancer.
  • cancer describes any array of different diseases linked by cumulative multiple genetic mutations, which result in the activation of oncogenes and/or the inactivation of tumor suppressor genes and/or linked by uncontrolled cellular proliferation. The cause and source of these mutations differs between different cancers of human body organs.
  • a brain cancer or tumor may be a glioma or non-glioma brain tumor.
  • the term “cancer” and “tumor” may be used interchangeably herein.
  • “cancer” may include any one of the following states: glioma, adenoma, blastoma, carcinoma, sarcoma and inclusive of any one of Medulloblastoma, Ependymoma, Astrocytoma, Optical nerve glioma, Brain stem glioma, Oligodendroglioma, Gangliogliomas, Craniopharyngioma or Pineal Region Tumors.
  • Reference to a "glioma” includes GMB, astrocytoma and anaplastic astrocytoma or related brain cancers.
  • Tau protein is an important protein as it is the protein expressed in the central nervous system and plays a critical role in the neuronal architecture by stabilizing intracellular microtubule network. Accordingly any impairment of the physiological role of the tau protein either by truncation, hyper-phosphorylation or by disturbing the balance between the six naturally occurring tau isoforms is detrimental to the subject and leads to the formation of neurofibrillary tangles (NFT), dystrophic neurites and neuropil threads.
  • NFT neurofibrillary tangles
  • the major protein subunit of these structures is microtubule associated protein tau.
  • the amount of NFT found in autopsies of AD patients correlates with clinical symptoms including intellectual decline.
  • tau protein plays a critical role in AD pathology.
  • the recent discovery of cosegregation of specific mutations in the tau gene with the disease frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) has confirmed that certain abnormalities in the tau protein can be a primary cause of neurodegeneration and dementia in affected individuals.
  • the activity of the complexes described herein to reduce levels of tau phosphorylation is as a result of their ability to deliver metal to cells and hence their anti-oxidant activity. It is felt that the ability of the complexes to act as anti-oxidants mean that they provide protection from OS which is desirable as OS can lead to hyper-phosphorylation of tau and cell dysfunction. As a consequence the ability of these complexes to deliver biologically important metals to cells allows them to function as anti-oxidants (especially where the oxidative stress is caused by metal deficiency) which in turn means the metal complexes may have the ability to prevent (or treat) tau-opathies.
  • disorders or conditions that are recognized as being tau disorders or more colloquially Tauopathies.
  • Disorders of this type include Richardson's syndrome, Progressive Supranuclear Palsy, Agryrophilic grain disease, corticobasal degeneration, Pick's disease, frontotemporar dementia linked with parkinsonism linked to chromosome 17 9FTDP-17), post-encephalitic parkinsonism (PEP), dementia pugilistica, Down's syndrome, Alzheimers disease, Familial British dementia, Familial Danish dementia, Parkinsons' disease, Parkinsons Disease complex of Guam (PDC), myotonic dystrophy, Hallevorden-Spatz disease, and Niemann-Pick type C.
  • the complexes may also be used in the treatment of an Abeta related disorder.
  • Abeta disorders are known including disorders selected from the group consisting of Parkinson's disease, Alzheimer's disease, Multiple sclerosis, Neuropathies, Huntington's disease, Prion disease, motor neurone disease, Amyotrophic lateral sclerosis (ALS), Menke's disease, and amyloidoses.
  • MMPs matrix metallo-proteinases
  • MMPs matrix metalloproteinases
  • Administration of complexes within Formula (I) to humans can be by any of the accepted modes of administration well known in the art. For example they may be administered by enteral administration such as oral or rectal, or by parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal routes. Injection can be bolus or via constant or intermittent infusion.
  • the active complex is typically included in a pharmaceutically acceptable carrier or diluent and in an amount sufficient to deliver to the subject a therapeutically effective dose.
  • the complexes of the invention can be administered in any form or mode which makes the complex bio-available.
  • One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the complex selected, the condition to be treated, the stage of the condition to be treated and other relevant circumstances. We refer the reader to Remingtons Pharmaceutical Sciences, 19th edition, Mach Publishing Co. (1995 ) for further information.
  • the complexes of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, diluent or excipient.
  • the complexes are, however, typically used in the form of pharmaceutical compositions which are formulated depending on the desired mode of administration.
  • the present invention provides a pharmaceutical composition including a complex of Formula (I) and a pharmaceutically acceptable carrier, diluent or excipient.
  • the compositions are prepared in manners well known in the art.
  • kits can include a composition including an effective agent either as concentrates (including lyophilized compositions), which can be diluted further prior to use or they can be provided at the concentration of use, where the vials may include one or more dosages.
  • single dosages can be provided in sterile vials so that the physician can employ the vials directly, where the vials will have the desired amount and concentration of agent(s).
  • Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the complexes of the invention may be used or administered in combination with one or more additional drug (s) that are useful for the treatment of the disorder/diseases mentioned.
  • the components can be administered in the same formulation or in separate formulations. If administered in separate formulations the complexes of the invention may be administered sequentially or simultaneously with the other drug(s).
  • compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin.
  • the complexes can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active complex is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and gly
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • the complexes can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • the active complexes can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring, and perfuming agents.
  • Suspensions in addition to the active complexes, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the complexes of this invention with suitable nonirritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active complex.
  • suitable nonirritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active complex.
  • Dosage forms for topical administration of a complex of this invention include powders, patches, sprays, ointments and inhalants.
  • the active complex is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.
  • the amount of complex administered will preferably treat and reduce or alleviate the condition.
  • a therapeutically effective amount can be readily determined by an attending diagnostician by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount a number of factors are to be considered including but not limited to, the species of animal, its size, age and general health, the specific condition involved, the severity of the condition, the response of the subject to treatment, the particular complex administered, the mode of administration, the bioavailability of the preparation administered, the dose regime selected, the use of other medications and other relevant circumstances.
  • a preferred dosage will be a range from about 0.01 to 300 mg per kilogram of body weight per day.
  • a more preferred dosage will be in the range from 0.1 to 100 mg per kilogram of body weight per day, more preferably from 0.2 to 80 mg per kilogram of body weight per day, even more preferably 0.2 to 50 mg per kilogram of body weight per day.
  • a suitable dose can be administered in multiple sub-doses per day.
  • the complexes of the various embodiments may be prepared using the reaction routes and synthesis schemes as described below, employing the techniques available in the art for each of the individual step/reactions and using starting materials that are readily available.
  • the preparation of particular complexes of the embodiments is described in detail in the following examples, but the artisan will recognize that the chemical reactions described may be readily adapted to prepare a number of other agents of the various embodiments.
  • the synthesis of non-exemplified complexes may be successfully performed by modifications apparent to those skilled in the art, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions.
  • a list of suitable protecting groups in organic synthesis can be found in T.W. Greene's Protective Groups in Organic Synthesis, John Wiley & Sons, 1981 .
  • other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other complexes of the various embodiments.
  • Reagents useful for synthesizing compounds may be obtained or prepared according to techniques known in the art.
  • THF Tetrahydrofuran
  • DMF N,N-dimethylformamide
  • Nuclear magnetic resonance spectra (NMR) spectra were acquired with either a Varian 400 MHz spectrometer ( 1 H at 400 MHz) or a Varian Inova 500NMR ( 1 H at 500 MHz). All chemical shifts were referenced to residual solvent peaks and are quoted in ppm relative to TMS. All spectra were recorded in d 6 -DMSO. Mass spectra were recorded using the electrospray technique (positive ion) VG BioQ Triple Quadrupole Mass Spectrometer. All reagents and other solvents were obtained from standard commercial sources and were used as received. ATSMH 2 , [Cu(ATSM)], [Zn(ATSM)], ATSPH 2 , [Cu(ATSP], [Zn(ATSP)].
  • ATSEH 2 [Cu(ATSE)], GTSMH 2 and [Cu(GTSM)] were prepared by variations of reported procedures, see: 1) P. J. Blower, T. C. Castle, A. R. Cowley, J. R. Dilworth, P. S. Donnelly, E. Labisbal, F. E. Sowrey, S. J. Teat and M. J. Went, Dalton Trans., 2003, 4416-4425 and references therein; 2) J. L. J. Dearling, J. S. Lewis, G. D. Mullen, M. J. Welch, and P. J. Blower, J. Biol. Inorg. Chem., 2002, 7, 249 and references therein; 3) P. McQuade, K. E.
  • ATSEH 2 (0.134 g, 0.46 mmol) and Zn(CH 3 CO 2 ) 2 .2H 2 O (0.102 g, 0.46 mmol) were added to ethanol (5 mL). The mixture was heated at reflux for 2 hours under an atmosphere of nitrogen and then allowed to cool to room temperature. The bright yellow solid that formed was collected by filtration and washed with ethanol, and diethyl ether to give [Zn(ATSE)] as a yellow powder (0.122 g, 0.35 mmol, 76 %).
  • 1,2-Cyclohexanedione (0.439 g, 3.92 mmol) was added to ethanol (25 mL) followed by N 4-ethyl-3-thiosemicarbazide (0.933 g, 7.83 mmol) and a few drops of H 2 SO 4 (conc). The mixture was heated at reflux under an atmosphere of nitrogen for 3 hours and then allowed to cool to room temperature. A yellow precipitate formed which was collected by filtration and washed with ethanol, and diethyl ether to ChexTSE as a yellow solid (0.945 g, 3.00 mmol, 76 %).
  • ChexTSE (0.190 g, 0.60 mmol) was added to ethanol (10 mL) followed by Zn(CH 3 CO 2 ).2H 2 O (0.133 g, 0.60 mmol) and the mixture was heated at reflux under an atmosphere of nitrogen for 3 hours. The mixture was allowed to cool to room temperature and a yellow solid precipitated. The solid was collected by filtration and washed with ethanol, and diethyl ether to give [Zn(Chextsc)] as a yellow solid (0.157 g, 0.42 mmol, 70 %).
  • Zinc(II) acetate dehydrate (0.03 g, 0.14 mmol) was added to a stirred solution of the ligand ( 3 ) (0.06 g, 0.14 mmol) dissolved in minimal DMF (1 mL). An orange colour change occurred immediately.
  • the reaction was stirred under an argon atmosphere at room temperature for 1 h, and then concentrated to an orange gum. The gum was sonicated in ethanol (2 mL) causing a bright orange solid to precipitate out of solution. The solid was removed and washed with hot ethanol (2 x 1 mL), ether (2 ml) and air dried to afford the zinc complex as a orange solid (0.04 g, 0.08 mmol, 62%).
  • Step 1 acetal protected mono substituted (methylthiosemicarbazide) ligand ( XX ).
  • Step 2 Formation of the aldehyde ( XXI ).
  • Step 3 Reaction with second thiosemicarbazides-unsymmetrical ligand formation ( XXII ).
  • Step 4 Unsymmetrical GTS copper complex formation ( XXIII ). A19
  • Zinc(II) acetate dihydrate (0.05 g, 0.24 mmol) was added to a stirred solution of the ligand ( 12 ) (0.07 g, 0.24 mmol) dissolved in minimal DMF (1 mL). An orange colour change occurred immediately. The reaction was stirred under an argon atmosphere at room temperature for 1 h, and then concentrated to an orange gum. The gum was sonicated in ethanol (1 mL) and the solid was removed and washed with ether (1 ml) and air dried to afford the zinc complex as an orange solid (0.04 g, 0.11 mmol, 47%).
  • Table 2 Complex No Structure A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24
  • APP-transfected Chinese Hamster Ovary (APP-CHO) cells were treated with a range of [Cu(BTSC] complexes with dialkyl backbones at 1-50 ⁇ M for 6 hr in serum-free culture medium. Cells were washed twice with phosphate buffered saline (PBS) and cells scraped into fresh PBS and pelleted at 10,000 rpm in a microfuge for 5 min. Supernatant was discarded and cell pellets frozen at -70°C until metal levels were determined by inductively-coupled plasma mass spectrometry (ICP-MS) at the Department of Pathology, University of Melbourne.
  • ICP-MS inductively-coupled plasma mass spectrometry
  • Treatment of the cells with [Zn(BTSC)] complexes resulted in significant increases in the intracellular Zn levels as measured by ICP-MS.
  • [Zn(ATSM)] and [Zn(ATSE)] induced 8.2 ⁇ 0.25 and 9.8 ⁇ 0.9 fold increases in cellular Zn levels respectively ( Figure 2 ).
  • APP-transfected CHO cells were treated with [Zn(BTSC)] complexes at 1-50 ⁇ M for 6 hr in serum-free culture medium.
  • APP-CHO cells with [Cu(GTSM)] resulted in an increase in the intracellular copper levels as expected of the cell permeable Cu-ligand.
  • Five treatment regimes were used namely a control, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 25 ⁇ M and 50 ⁇ M ( Figure 3 ).
  • APP-transfected CHO cells were treated with each of the doses of [Cu(GTSM)] for 6 hr in serum-free medium and conditioned medium was then collected and assayed for A ⁇ 1-40 peptide by routine A ⁇ ELISA.
  • [Cu(GTSM)] significantly inhibited A ⁇ 1-40 levels in the medium at all concentrations tested when compared to uncomplexed GTSM.
  • APP-CHO cells with a number of zinc complexes resulted in an increase in the intracellular zinc levels as expected of the cell permeable Zn-ligand.
  • Five treatment regimes were used namely a control, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 25 ⁇ M and 50 ⁇ M ( Figure 4 ).
  • APP-transfected CHO cells were treated with each of the doses of [Zn(BTSC)] for 6 hr in serum-free medium and conditioned medium was then collected and assayed for A ⁇ 1-40 peptide by routine A ⁇ ELISA. All Zn-BTSC significantly inhibited A ⁇ 1-40 levels in the medium at 10-50 ⁇ M.
  • Zn-Chex TSE and Zn-ATSE also inhibited A ⁇ at 1 ⁇ M.
  • Example 13 Cu inhibits Zn uptake and loss of secreted A ⁇ induced by [Zn(BTSC)]
  • APP-CHO cells were generated by expressing the 695 amino acid APP cDNA in the pIRESpuro2 expression vector (Clontech, Mountain View, California, USA). Cells were transfected using Lipofectamine 2000 and cultured in RPMI-1640 media supplemented with 1mM glutamine and 10% fetal bovine serum (all from Invitrogen, Mount Waverley, Victoria, Australia). Transfected cells were selected and maintained using 7.5 ⁇ g/ml puromycin (Sigma-Aldrich).
  • APP-CHO cells were passaged at a ratio of 1:5 and grown in 6 well plates for 3 days before experiments. Compounds were prepared as a 10 mM stock solution in DMSO and added to serum-free RPMI medium supplemented with puromycin. Medium was briefly mixed by aspiration prior to addition to cells. Control cultures were treated with vehicle (DMSO) alone. Cultures were incubated for 6 hr and conditioned media taken for measurement of A ⁇ 1-40 levels by ELISA.
  • a ⁇ levels were determined in culture medium using the 384 well A ⁇ 1-40 ELISA protocol.
  • 384 well plates were coated with monoclonal antibody (mAb) G2-10 in carbonate-bicarbonate coating buffer (pH 9.6) for A ⁇ 1-40 detection. The plates were left to incubate overnight at 4°C with rocking. The plates were then washed three times with PBST at RT with rocking and the solution discarded after each wash. Then 100 ⁇ L of 0.5% (w/v) hydrolysed casein in PBS (pH 7.4) was added to each well and left to incubate for 2 hr at 37°C to prevent non-specific binding. The plates were then washed three times with PBST at RT with rocking.
  • M17 human neuroblastoma cells were plated out on 6 well plates and left overnight. Enough cells were added to give approximately 70 % confluent the following day of the experiment.
  • test cells were incubated in 1 ml of media and compound mix for 5 hours at 37°C. At the end of the incubation the media was removed with a vacuum aspirator and 1 ml of PBS added to dislodge the cells. Cells are then put into Eppendorf tubes and pelleted. The PBS is removed and the remaining cell pellets are frozen at -20 C.
  • the instrument was calibrated using Blank, 10, 50 and 100 ppb of a certified multi-element ICPMS standard solution (ICP-MS- CAI2-1, Accustandard) for Fe, Cu and Zn in 1% nitric acid. Used an certified internal standard solution containing 100 ppb Yttrium (Y 89) as an internal control (ICP-MS- IS-MIX1-1, Accustandard).
  • test cells were cultured at 37°C/5% CO 2 till almost confluent in 75 cm 2 flask.
  • the media was removed and the cells incubated with 5 ml PBS for about 5 mins to dislodge cells from the plastic surface.
  • a pipette was used to re-suspend cells and 5 mls of growth media added.
  • the cell suspension was removed and added to 15 ml Falcon tube. The suspension was mixed well by inversion and about 100 ⁇ l transferred into an Eppendorf.
  • a typical assay assessing 15 compounds uses five 48 well plates.
  • the inner 24 wells are the only ones used to reduce the amount of evaporation over 48 hrs. 200 ⁇ l of media was added to each of the inner 24 wells. Cell suspensions were mixed by inversion and the desired number of cells added to each well. Cell addition was continued across each plate and the the cell suspension mixed in the falcon tube by inversion between each plate. The plates were given a minor shake and returned to a 37 °C incubator. Plates were left overnight for cells to settle in the wells.
  • DMSO was added to the Eppendorfs (typically 200-500 ⁇ l), vortexed until dissolved and incubated with the compounds at 37 °C for 60 mins to aid solubilisation. Compounds were removed and again vortexed and checked for any undissolved compound.
  • the 10 mM stock solutions should then be diluted 1:10 to make a final concentration of 1 mM.
  • 180 ⁇ l of DMSO was added to the test tube and 20 ⁇ l of each of the compound solutions was added to each of the test tubes to create the test solutions which were then vortexed again to ensure complete homogenation of the test mixture.
  • Each compound is then diluted to final concentrations of 10 ⁇ M and 1 ⁇ M.
  • the desired amount of the test solution and the control sample was added to the plates which were then returned to the incubator for a 48 hour period at 37°C.
  • the plates were removed from the incubator and an aspirator used to remove the media from the first plate.
  • 220 ⁇ l of MTT/media solution was added to each well. Plates were then returned to 37 °C and incubated for 1 hour. After 1 hour the plates were removed from the incubator and the media/MTT solution removed using the aspirator vacuum pump.
  • MTT is a tetrazolium salt which is converted from yellow to purple by active mitochondria. The more cells present and therefore more mitochondria results in a more intense purple colour.
  • the plates can now be read on a plate reader at 570 nm. The results are provided in Table 4.
  • the neuronal cells were allowed to mature for 6 days in culture before commencing treatment using freshly prepared modified culture media (neurobasal medium plus B27 supplements (minus antioxidants), geneticin, and cytosine ⁇ -D-arabinofuranoside (Sigma)).
  • modified culture media neutral medium plus B27 supplements (minus antioxidants), geneticin, and cytosine ⁇ -D-arabinofuranoside (Sigma)
  • test compound stock solutions were diluted to the final concentration (as outlined below) in 200 ⁇ L modified culture media.
  • the mixtures were then added to neuronal cells for up to 4 days.
  • the health of the cells was periodically monitored by phase contrast microscopy, and cell viability was quantitated using the MTS assay (Promega, Madison, WI).
  • the experimental media was replaced with freshly prepared modified culture media containing 10% v/v MTS.
  • the plates were returned to the 37 °C incubated with 5% CO 2 for 3 h. Then a 150- ⁇ L aliquot from each well was transferred to separate wells of a 96-well plate. The color change of each well was determined by measuring the absorbance at 490 nm and background readings of MTS incubated in cell-free medium were subtracted from each value before calculations. The data were normalized and calculated as a percentage of untreated vehicle control values. Data are shown as mean ⁇ S.E. All samples are tested in triplicate and untreated vehicle treated cells were present on every plate tested.
  • Test compounds were initially dissolved in 100% DMSO (Sigma) to a concentration of 5 mM. 5 mM stock solution was diluted to give working concentrations of 1 mM and 0.1 mM. Test compounds were not added directly to cells, instead they were added to a sterile 48 well 'Drug Plate', then diluted with modified culture media. After the addition of the modified culture media to the drug plate, the media was mixed gently and 200 ⁇ l aliquots (in triplicates) was added to plates containing neurons.
  • a representative complex (A8) was subjected to an OxyblotTM assay.
  • This assay detects carbonyl groups (aldehydes and ketones) on proteins produced by metal-catalysed oxidation. Proteins are sourced from brain slices of Tg mice that have been subject to the test compounds. Carbonyl groups are thought to be a 'hallmark of the oxidation status of the proteins. Carbonyl groups on protein side-chains are derivatised to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH). The samples can then undergo gel electrophoresis or directly put on a dot-blot. Primary antibody detects the DNP exposed moiety of proteins, using a provided standard to read off particular concentrations of DNP found in the proteins.
  • the assay was performed as follows:
  • the dot-blotting device had a 96-well plate installed to collect the sample on the bottom, and a pre-soaked (PBS) nitrocellulose membrane (14cmX9cm) fitted and sealed between the soft plastic thin layer and the topmost hard plastic layer.
  • PBS pre-soaked nitrocellulose membrane
  • the membrane was placed in blocking buffer (40ml) for 1 hr, rinsed twice briefly with PBS-T. Blot was then incubated with primary antibody from the kit (rabbit anti-DNP; 1:150 dilution in PBS-T, 134 ⁇ l antibody in ⁇ 20 ⁇ l PBS-T) for one hour, then washed twice immediately, followed by 2X15min and 2X5min washes with PBS-T. Further incubated with secondary anitbody 1:300, 67 ⁇ l in 20 ⁇ l (goat anti-rabbit IgG-HRP) then rinsed again as described for post-primary antibody step. Blot was developed by incubating in chemiluminescence detection reagent (GE) 4ml reagent1 : 4ml reagent 2 for 2 min.
  • GE chemiluminescence detection reagent
  • the RHS of the brain including the cerebellum was collected, recorded the wet weight and placed in a Beckman labelled ultracentrifuge tube. This brain sample is used to test the levels of Abeta and metal in the Supernatant (SN) and Pellet homogenate.
  • SN Supernatant
  • a 20 ul aliquot of Pellet and SN were diluted 1:10, i.e. added 180 ⁇ l of PBS. Determined protein concentration as per BCA assay method. The Absorbance levels were around the 0.3-0.9 Abs. Using the excel program determined the volume of PBS required to make a stock solution of: 3 ⁇ g/ ⁇ l of protein of SN and a 0.2 ⁇ g/ ⁇ l of protein of Pellet using the 20 ⁇ l aliquots.
  • rabbit HRP DAKO
  • Washed blots for 6 x 10 min with TBST used ECL reagent (Amersham) to develop blots (8ml per blot, 1:1 ratio reagent A and B). Incubated the blots in ECL for 2 min.
  • Non transgenic (non-Tg) (BL/6XSJL) Tg APP2576 (-) female mice aged -12-14 months were selected fro the bioavailability trial.
  • mice were orally administered either 5 or 30 mg/kg.
  • mice received the regular drug dose spiked with the radiolabelled Cu-64 or Zn-65 analogue.
  • Radiolabeled compounds were prepared by standard protocols (P. S. Donnelly, O. Golovko, J. M. Heslop, P. Burke, J. C. Clark, J. R. Dilworth, F. I. Aigbirhio, J. Label. Compd. Radiopharm., 2005, 48 , S163).
  • Bioavailability of the drug was given by measuring radioactivity of tissue samples. Duration: Compounds were orally administered by gavage for 7 or 8 consecutive days Vehicle: SSV pH6.3
  • Drug preparation Dosage volume set at 100 ⁇ l for a 25g mouse or 4 times the mouse weight. Drug dosage is 5mg/kg.
  • the results of the bioavailability study are provided in Table 12 with a results summary being given in Table 13.
  • Table 12 Mouse Cmpd Blood (CPM) Liver (CPM) (Brain CPM) Blood (Vol) Liver (g) Brain (g) 1 A8 7925 38985 516 1.0 0.27 0.45 2 A8 6932 46158 486 1.0 0.37 0.30 3 A8 34644 170710 1998 1.0 0.43 0.22 4 A8 11363 78729 952 1.0 0.29 0.40 5 A16 4626 122655 966 1.0 0.38 0.49 6 A16 356 2892 162 1.0 0.21 0.37 7 A16 5908 81209 627 1.0 0.24 0.37 8 A16 5664 84918 773 1.0 0.20 0.42 9 A23 5643 99907 1084 1.0 0.21 0.37 10 A23 5242 56261 7667
  • Example 23 BTSC-metal complexes activate PI3K and JNK-dependent pathways resulting in increased degradation of secreted A ⁇ 1-40.
  • APP-CHO cells were treated with 10 ⁇ M metal-BTSC complexes for 6 hr.
  • Cells were extracted into Phosphosafe (Novagen) lysis buffer and proteins separated by gel-electrophoresis on 12% tris-glycine gels. Proteins were transferred to PVDF membranes and blocked with milk powder for 1 hr. Blots were then probed for total and phosphorylated forms of Akt, JNK and GSK3 using antibodies from Cell Signaling Technologies. After detection with secondary antisera (HRP-labelled), blots were analysed for signals using chemiluminescence analysis on a GeneGnome image scanner. Activation of PI3K is shown by increased levels of phosphorylated Akt (p-Akt). Activated JNk is shown by increased p-JNK. Increased levels of deactivated GSK3 is shown by increased p-GSK3.
  • APP-CHO cells were treated with 10 ⁇ M [Zn(ATSE)] with or without specific inhibitors of PI3K (LY294002), JNK (SP600125) or p38 (SB203580) (25 ⁇ M of each) for 6 hr and conditioned medium analysed for levels of A ⁇ 1-40 by routine ELISA. Inhibition of JNK by SP600125 prevented the loss of A ⁇ by [Zn(ATSE)]. Inhibition of PI3k by LY294002 prevented the loss of A ⁇ by [Zn(ATSE)].
  • a Y maze was set up with the arms labelled. The floor of the maze was covered with sawdust evenly in all 3 arms (used saw-dust for black mice and fibre-cycle for agouti mice. Each mouse to be used in the trail was then randomly allocated a starting and blocked arm.
  • the video data was then analysed computationally to determine the period that each mouse spent in each arm of the maze in the first incursion and in the second incursion.
  • mice double transgenic APP/PS1 mice were split randomly into two groups of 12. Mice in each group. The first group was subjected to a control or sham dosage whereas the second group was treated with CuGTSM at a dosage of 10mg/kg.. The results are shown in table 14. Table 14 Y-Maze Data Treatment % Visit to Novel arm % Visit to Start Arm % Visit to Other Arm Control 33.2 30.7 36.1 CuGTSM/10mg/kg. 47.9 29.7 22.4
  • mice In normal mice the normal response is that upon being re-trialled (i.e with the novel arm un-blocked) mice will spend a significantly greater proportion of time in the novel arm in comparison with the start arm and the other arm. Thus typically the mice will spend their time 50%, 25%, 25%. In contrast if the percentage of time or number of visits is around equal the results are interpreted that the mice had no memory of the initial experience and hence did not recognize the novel arm as such. The above results therefore clearly indicate that the mice treated with CuGTSM had significant memory improvement in comparison with the control mouse.
  • M17 (human) or N2a (murine) neuroblastoma cells were plated at a passaging ratio of 1:4 from a 90% confluent flask of cells and grown until 80-90% confluent.
  • Medium was replaced with fresh serum-free OptiMem and cells were exposed to 1 or 100 nM CuGTSM (from 10 mM stock in DMSO) for 18 hr (overnight).
  • Medium was removed and cells extracted into Phosphosafe (Invitrogen) extraction buffer and frozen at -80oC.
  • Western blots were performed on cell lysates for total GSK3 ⁇ and phospho-GSK3 ⁇ / ⁇ (Cell Signaling Technology).
  • the cell line was maintained in OPTI-MEM (Gibco) supplemented with 10 % foetal calf serum (FCS), Non-essential amino acids, sodium pyruvate and Penn/Strep. Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5 % CO 2 . Cell assays were plated out into 48 well culture plates at 4 x 10 4 cells per well. Cells were left to settle overnight then incubated with drugs for 24h prior to being subjected to MTT assays for cell viability.
  • FCS foetal calf serum
  • the viability of cells was assessed using an MTT assay. Active mitochondria convert the yellow MTT tetrazolium salt to purple formazan which can be measured by a spectrophotometer as described in methods previously (Mossman 1983).
  • the mixture of media and reagents e.g. Dopamine was aspirated off and media containing 5 mg/ml MTT is added and the plate incubated for 1 h at 37°C. The remaining media/MTT solution is aspirated off and the cells were then solubilised with DMSO. The 48 well plate is then read at 595 nm on a plate reader.
  • a partial lesion of the SNpc was produced in the mice by injecting the neurotoxin 6-OHDA into the right SNpc.
  • the mouse was injected with atropine (0.5 mg/kg) to reduce respiratory tract secretions together with xylazine (10 mg/kg) to produce sedation (intramuscular injection with a 27 gauge needle, 60 microlitres).
  • Mice were anesthetized with 4% chloral hydrate in PBS (10 ml/kg, i.p.), and heads were secured in a stereotaxic head frame with the bite bar 3 mm above horizontal.
  • a 1.65 mg/ml solution of 6-OHDA was prepared with ascorbic acid (0.2 mg/ml) and kept on ice until the time of injection.
  • the needle was left in place for 5 min then slowly withdrawn at a rate of 1 mm/min.
  • Test drugs were suspended in standard suspension vehicle (SSV; NaCl 0.9 % w/v, Na-CMC 0.05 % w/v, Benzyl alcohol 0.05 % v/v, Tween 80 0.04 % v/v) and were delivered by oral gavage at a daily dosage of 10 or 30 mg/kg for 7 days pre lesion and then a period of 14 consecutive days post lesion (Cherny et al., 2001); controls received SSV alone.
  • SSV standard suspension vehicle
  • Rotation-this assay is only applicable to mice and rats that receive a unilateral injection of 6-OHDA to produce a partial lesion of nigral neurons.
  • the lesioned rodent is placed in a bowl and videotaped for one hour, then injected with 5 mg/kg amphetamine Fourteen days after lesioning the mouse was connected to the automated Rotacount system (Columbus Instruments, Columbus, OH, USA). This records contralateral and ipsilateral rotations.
  • the mouse is attached so that any movement clockwise or anticlockwise is registered by a sensor that is then monitored and counted by a computer (SOFTWARE). Once connected baseline movement levels are established by leaving the rodent for 30 mins.
  • the mice are then injected via an intraperitoneal injection of 5 mg/kg amphetamine (Sigma) and then the movement recorded for a further hour. Data from the sensors is then plotted and analysed .
  • mice were killed by an overdose of sodium pentobarbitone (Lethobarb; 0.35 mg/gm) and perfused with 30 ml of warmed (37°C) 0.1 M PBS, pH 7.4, with heparin (1 ⁇ ml), followed by 30 ml of chilled 4% paraformaldehyde (Sigma, St. Louis, MO) and 0.2% picric acid in 0.1 M phosphate buffer (4°C), pH 7.4. The brains were then removed and left at 4°C overnight in 30% sucrose in PBS.
  • Lethobarb 0.35 mg/gm
  • Sections were fixed in 100% ethanol for 15 min at 4C. The sections were then air dried and rinsed with 0.1 M PBS before being incubated in blocking solution (3 % NGS, 0.3 % (v/v) Triton, 0.1 M PBS) for 15 min at RT. After 3 5 min washes with 0.1 M PBS the sections were incubated overnight at RT with rabbit anti-TH antibody (1:800 chemicon) in 1 % NGS/ 0.3 % Triton/0.1 M PBS. This was followed by a further washing step and by incubation of goat anti-rabbit IgG (1:300 SOURCE) in 1% NGS/0.3% Triton/0.1M PBS for 2 h at RT.
  • blocking solution 3 % NGS, 0.3 % (v/v) Triton, 0.1 M PBS
  • reaction was visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB) for 15 min and then DAB with hydrogen peroxide for 5 min. Section are washed three times in 0.1M PBS for 10 min and then counterstained with Neutral Red (50 s).
  • DAB 3,3-diaminobenzidine tetrahydrochloride
  • the total number of DA neurons in the SN were estimated using a fractionator sampling design [refs Finkelstein et al 2000 , Stanic et al 2004 and West and Gendersen 1990].
  • Mice brains are sectioned in a 1:3 series at 40 ⁇ m and immunohistochemistry performed for Nissl stained sections. Counts are made at regular predetermined intervals (x 140 um, y 140 um). Systematic samples of the area occupied by the nuclei are made from a random starting point. An unbiased counting frame of known area (45 um x 35 um) is superimposed on the image of the tissue sections.
  • the cells were plated onto 6 well plate and grown to ⁇ 70% confluency (maximum) before treatment.
  • the OptiMEM media was then removed from cells and replaced with various concentrations of Cu (GTSM), 1 mL to each well.
  • GTSM concentrations of Cu
  • the cells are treated for 6 hours at 37°C and following the 6 hour treatment, the cells were harvested for western blot analysis using a cell lysis protocol.
  • the cells were plated onto 6 well plate and grown to ⁇ 90% confluency before treatment.
  • the DMEM media was removed from cells and replaced with various concentrations of Cu (GTSM), 1mL to each well.
  • GTSM concentrations of Cu
  • the cells are treated for 6 hours at 37°C. Following the 6 hour treatment, the cells were harvested and protein lysates were prepared using a cell lysis protocol.
  • SSV Standard Suspension Vehicle
  • composition Conc 1 L 500 ml NaCl 0.9% (w/v) 9.0 g 4.5 g Na-CMC 0.5% (w/v) 5.0 g 2.5 g Benzyl alcohol 0.5% (v/v) 5.0 ml 2.5 ml Tween 80 0.4% (v/v) 4.0 ml 2.0 ml
  • CuATSM in dosage given to mice was 30mg/kg through gavaging. It is stored at -20°C and thawed on the day of administration.
  • mice 4X mouse weight. (eg. 25g mouse received 100 ⁇ l of suspension).
  • Cu-ATSM was administered once daily (5 days/week) in TgSOD1 G93A mice until they reach end stage (lost of 15-20% body weight, one hindlimb paralysis, and loss of partial motor function). The animal is culled at this point and the survival time plotted. The results are shown in figure 22 .
  • Transfected U87MG-EGFR cells were treated with 25 ⁇ M CuGTSM or DMSO as vehicle control for 5 hr and EGFR phosphorylation (tyr1068) determined by Western blot.
  • Antisera to EGFRtyr1068 was routinely used as this is one of the critical tyrosine residues involved in EGFR activation and downstream signalling. It is also routinely reported in the literature on EGFR activation.
  • Figure 24A shows that EGFR phosphorylation and therefore activation occurred on treatment with CuGTSM compared to vehicle control.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Neurosurgery (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Psychology (AREA)
  • Psychiatry (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Communicable Diseases (AREA)
  • Pain & Pain Management (AREA)
  • Vascular Medicine (AREA)
  • Urology & Nephrology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Claims (6)

  1. Complexe métallique de Formule (I) destiné à être utilisé dans le traitement de la maladie de Parkinson ou de la sclérose latérale amyotrophique,
    Figure imgb0128
     où :
    le complexe est symétrique ;
    M est Cu ;
    R1 et R5 sont chacun un hydrogène ;
    R2 et R6 sont chacun un méthyle, éthyle, ou phényle ; et
    R3 et R4 sont chacun un méthyle.
  2. Complexe selon la revendication 1 destiné à être utilisé dans le traitement de la maladie de Parkinson.
  3. Complexe selon la revendication 1 destiné à être utilisé dans le traitement de la sclérose latérale amyotrophique.
  4. Complexe destiné à être utilisé dans le traitement selon l'une quelconque des revendications 1 à 3, où R2 et R6 sont chacun un méthyle.
  5. Complexe destiné à être utilisé dans le traitement selon l'une quelconque des revendications 1 à 3, où R2 et R6 sont chacun un éthyle.
  6. Complexe destiné à être utilisé dans le traitement selon l'une quelconque des revendications 1 à 3, où R2 et R6 sont chacun un phényle.
EP14166833.5A 2006-11-20 2007-11-20 Agents d'apport métallique et leur utilisation thérapeutique Active EP2813223B8 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP17170883.7A EP3238719A1 (fr) 2006-11-20 2007-11-20 Agents d'apport métallique pour le traitment de la sclérose en plaques

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US85992106P 2006-11-20 2006-11-20
EP07815594A EP2099441A4 (fr) 2006-11-20 2007-11-20 Agents de délivrance de métaux et leurs utilisations thérapeutiques

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
EP07815594A Division EP2099441A4 (fr) 2006-11-20 2007-11-20 Agents de délivrance de métaux et leurs utilisations thérapeutiques

Related Child Applications (2)

Application Number Title Priority Date Filing Date
EP17170883.7A Division-Into EP3238719A1 (fr) 2006-11-20 2007-11-20 Agents d'apport métallique pour le traitment de la sclérose en plaques
EP17170883.7A Division EP3238719A1 (fr) 2006-11-20 2007-11-20 Agents d'apport métallique pour le traitment de la sclérose en plaques

Publications (3)

Publication Number Publication Date
EP2813223A1 EP2813223A1 (fr) 2014-12-17
EP2813223B1 true EP2813223B1 (fr) 2017-10-25
EP2813223B8 EP2813223B8 (fr) 2018-03-07

Family

ID=39429309

Family Applications (3)

Application Number Title Priority Date Filing Date
EP17170883.7A Withdrawn EP3238719A1 (fr) 2006-11-20 2007-11-20 Agents d'apport métallique pour le traitment de la sclérose en plaques
EP07815594A Ceased EP2099441A4 (fr) 2006-11-20 2007-11-20 Agents de délivrance de métaux et leurs utilisations thérapeutiques
EP14166833.5A Active EP2813223B8 (fr) 2006-11-20 2007-11-20 Agents d'apport métallique et leur utilisation thérapeutique

Family Applications Before (2)

Application Number Title Priority Date Filing Date
EP17170883.7A Withdrawn EP3238719A1 (fr) 2006-11-20 2007-11-20 Agents d'apport métallique pour le traitment de la sclérose en plaques
EP07815594A Ceased EP2099441A4 (fr) 2006-11-20 2007-11-20 Agents de délivrance de métaux et leurs utilisations thérapeutiques

Country Status (5)

Country Link
US (2) US20120270850A1 (fr)
EP (3) EP3238719A1 (fr)
AU (1) AU2007324345B2 (fr)
ES (1) ES2650720T3 (fr)
WO (1) WO2008061306A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10413554B2 (en) 2006-11-20 2019-09-17 The University Of Melbourne Metal delivery agents and therapeutic uses of the same
ES2650720T3 (es) 2006-11-20 2018-01-22 The University Of Melbourne Agentes de suministro de metales y usos terapéuticos de los mismos
AU2009326867B2 (en) * 2008-12-12 2015-10-29 The University Of Melbourne Process for the preparation of asymmetrical bis(thiosemicarbazones)
JP6085810B2 (ja) * 2012-12-27 2017-03-01 日本メジフィジックス株式会社 放射性医薬及び医薬キット
WO2015070177A2 (fr) 2013-11-11 2015-05-14 Collaborative Medicinal Development, Llc Complexes métalliques et méthodes de traitement
US9422286B2 (en) 2014-01-27 2016-08-23 Washington University Metal-binding bifunctional compounds as diagnostic agents for Alzheimer's disease
TWI649300B (zh) * 2016-04-18 2019-02-01 南臺學校財團法人南臺科技大學 雙硫半卡腙配位體及其合成方法
US11142835B2 (en) 2016-06-10 2021-10-12 University Of Louisville Research Foundation, Inc. Compounds, their preparation, related compositions, catalysts, electrochemical cells, fuel cells, and uses thereof
US11208379B2 (en) 2017-05-04 2021-12-28 University Of Louisville Research Foundation, Inc. Compounds, compositions, methods for treating diseases, and methods for preparing compounds
WO2019046761A1 (fr) * 2017-09-01 2019-03-07 Oregon State University Complexes de métal thérapeutiques et ligands, leurs procédés de fabrication et d'utilisation
JP2022546700A (ja) * 2019-08-30 2022-11-07 リサーチ インスティチュート アット ネイションワイド チルドレンズ ホスピタル ミトコンドリア機能障害に関連する神経変性障害の治療のための銅－ａｔｓｍ
WO2021046092A1 (fr) * 2019-09-03 2021-03-11 Procypra Therapeutics, Llc Nouveau polymorphe et ses utilisations
EP4073046A4 (fr) * 2019-12-12 2024-02-21 The University of Melbourne Composés et méthodes de diagnostic, d'imagerie et de traitement de maladies et de troubles neurodégénératifs
WO2022047014A1 (fr) * 2020-08-26 2022-03-03 Als Therapy Development Institute Complexes de cuivre destinés au traitement d'états neurodégénératifs
CN113189318B (zh) * 2021-04-26 2022-05-10 杭州凯莱谱精准医疗检测技术有限公司 定量检测乳汁中微量元素的方法
CN116421594A (zh) * 2022-01-04 2023-07-14 中南大学 一种nlrp3炎性小体抑制剂及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10101566A (ja) * 1996-09-27 1998-04-21 Kagaku Gijutsu Shinko Jigyodan 網膜保護剤
GB0513812D0 (en) * 2005-07-05 2005-08-10 Isis Innovation Compounds for imaging and therapy
US7887803B2 (en) * 2005-12-02 2011-02-15 Amorfix Life Sciences Methods and compositions to treat misfolded-SOD1 mediated diseases
ES2650720T3 (es) 2006-11-20 2018-01-22 The University Of Melbourne Agentes de suministro de metales y usos terapéuticos de los mismos
WO2015070177A2 (fr) 2013-11-11 2015-05-14 Collaborative Medicinal Development, Llc Complexes métalliques et méthodes de traitement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
AU2007324345B2 (en) 2014-02-06
WO2008061306A1 (fr) 2008-05-29
AU2007324345A1 (en) 2008-05-29
US10159679B2 (en) 2018-12-25
EP2099441A1 (fr) 2009-09-16
EP3238719A1 (fr) 2017-11-01
US20120270850A1 (en) 2012-10-25
US20150105461A1 (en) 2015-04-16
EP2813223B8 (fr) 2018-03-07
ES2650720T3 (es) 2018-01-22
EP2099441A4 (fr) 2010-04-28
EP2813223A1 (fr) 2014-12-17

Similar Documents

Publication Publication Date Title
EP2813223B1 (fr) Agents d&#39;apport métallique et leur utilisation thérapeutique
US11123350B2 (en) Metal delivery agents and therapeutic uses of the same
EP3068762B1 (fr) Complexes métalliques et méthodes de traitement
Bolognin et al. Chelation therapy for neurodegenerative diseases
Armstrong et al. Comparative effects of metal chelating agents on the neuronal cytotoxicity induced by copper (Cu+ 2), iron (Fe+ 3) and zinc in the hippocampus
JP2019507796A (ja) イカリイン及びイカリチン誘導体
WO2014036379A2 (fr) Acides biliaires permettant de prévenir ou d&#39;arrêter la progression de la maladie de parkinson et de maladies neurodégénératives associées
JP7003045B2 (ja) 神経変性の治療のための方法及び医薬組成物
CN108697690B (zh) 苯并噻唑两亲物
AU2009288057B2 (en) 2, 4-disulfonyl pheny tert-butyl nitrone for the treatment of gliomas
Shin et al. Ceruloplasmin is an endogenous protectant against kainate neurotoxicity
Brembati et al. Alpha synuclein post translational modifications: potential targets for Parkinson’s disease therapy?
MX2012000303A (es) Composiciones farmaceuticas y metodos teraputicos que emplean una combinacion de un compuesto complejo de manganeso y una forma compleja no de manganeso del compuesto.
US20220289690A1 (en) Second generation inhibitors of mitochondrial permeability transition pore with improved plasma stability
Dhala et al. Chimeric Conjugates for Alzheimer’s Disease
US20140335019A1 (en) Compositions and methods for the treatment and analysis of neurological disorders
WO2009128050A2 (fr) Composition pharmaceutique et utilisation de celle-ci
US20220289587A1 (en) A novel polymorph and uses thereof
WO2017219087A1 (fr) Nouveaux composés de thiosemicarbazone et leurs analogues
WO2012139001A2 (fr) Inhibiteurs de liaison à une protéine kinase

Legal Events

Date Code Title Description
17P Request for examination filed

Effective date: 20140502

AC Divisional application: reference to earlier application

Ref document number: 2099441

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

R17P Request for examination filed (corrected)

Effective date: 20150610

RBV Designated contracting states (corrected)

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20160418

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20170515

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AC Divisional application: reference to earlier application

Ref document number: 2099441

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 939274

Country of ref document: AT

Kind code of ref document: T

Effective date: 20171115

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602007052843

Country of ref document: DE

GRAT Correction requested after decision to grant or after decision to maintain patent in amended form

Free format text: ORIGINAL CODE: EPIDOSNCDEC

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2650720

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20180122

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20171025

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 939274

Country of ref document: AT

Kind code of ref document: T

Effective date: 20171025

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 11

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180225

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180125

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180126

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602007052843

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20171120

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20171130

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20171120

26N No opposition filed

Effective date: 20180726

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 12

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20171120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20171130

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20071120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20171025

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20210930

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20211203

Year of fee payment: 15

Ref country code: FR

Payment date: 20211109

Year of fee payment: 15

Ref country code: DE

Payment date: 20210929

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20211012

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20221201

Year of fee payment: 16

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602007052843

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20221120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20221120

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20221120

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230601

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20221130

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20240102

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20221121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20221121

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231130